Rapid developments in the field of DNA microarrays have lead to a number of methods for synthetic preparation of DNA. Such methods include spotting pre-synthesized oligonucleotides, photolithography using mask or maskless techniques, in situ synthesis by printing reagents, and in situ parallel synthesis on a microarray of electrodes using electrochemical deblocking of protective groups. During electrochemical deblocking, a voltage applied to an electrode generates reagent that removes the protective group thus allowing continued synthesis. A review of oligonucleotide microarray synthesis is provided by: Gao, X., Gulari, E., Zhou, X., Biopolymers 2004, 73, 579. The synthetic preparation of a peptide array was originally reported in 1991 using photo-masking techniques. This method was extended in 2000 to include an addressable masking technique using photogenerated acids and/or in combination with photosensitizers for deblocking. Reviews of peptide microarray synthesis using photolabile deblocking are provided by: Pellois, P. J., Wang, W., Gao, X., J. Comb. Chem. 2000, 2, 355 and Fodor; S. P. A., Read, J. L., Pirrung, M. C., Stryer, L, Lu, A. T., Solas, D., Science, 1991, 251, 767. Some recent work using peptide arrays has utilized arrays produced by spotting pre-synthesized peptides or isolated proteins. A review of protein arrays is provided by: Cahill, D. J., Nordhoff, E. Adv. Biochem. Engin/Biotechnol. 2003, 83, 177.
During the synthesis of DNA or peptides on a microarray or other substrate, each successive addition of a respective monomer involves the removal of a protecting group to allow addition of the next monomer unit. In such a removal or deblocking step, a specific type of solution can be used that is commonly referred to as a deblocking solution, i.e., the solution deblocks the end of the chain of a DNA or peptide by removing a protective group to allow the addition of a next monomer unit. In general, protective groups can be acid-lable or base-labile, i.e., acidic conditions remove the acid-labile group and basic conditions remove the base-labile group. Additionally, some protecting groups are labile to only specific types of solvents. Alternatively, deblocking can be accomplished using photolabile-protecting groups, which can be removed by light of a certain wavelength. A review of photoremoveable protecting chemistry is provided by: Photoremovable Protecting Groups in Organic Chemistry, Pillai, V. N. R., Synthesis 39: 1-26 (1980). Use of protective groups is a common technique in organic synthesis and is used in the synthesis of DNA or peptides to control the addition points of successive units. Reviews of protective group chemistry are provided by: Protective Groups in Organic Synthesis, Greene, T. W. and Wuts, P. G. M., Wiley-Interscience, 1999 and Protecting Group Chemistry, Robertson, J., Oxford University Press, 2001.
Protecting groups can be removed by electrochemical methods on a microarray of electrodes as a step in the synthesis of polymers on the microarray. In this method, protecting groups are removed only at selected electrodes by applying a potential only at the selected electrodes. In order to prevent deprotection at neighboring electrodes, the method and the solution need to confine the electrochemical effects to the region immediately adjacent to the electrode undergoing deblocking. Crosstalk refers to the ability of a method and solution to substantially isolate deblocking to the active electrodes while substantially preventing deblocking outside of the active electrode area. Minimal crosstalk is desirable. Where an aqueous-based deblock solution having a buffer is used, the solution likely buffers the generation of acidic or basic species to the region near the electrode and prevents diffusion of such species to adjacent electrodes. However, in organic-based deblock solutions, the mechanism of preventing crosstalk is not necessarily well understood but may involve molecular interactions that remove or passify acidic reagent by some other species.
An aqueous-based deblock solution is disclosed in Montgomery, U.S. Pat. Nos. 6,093,302 and 6,280,595, the disclosures of which is incorporated by reference to the patents herein. In both Montgomery patents, a 0.10 M solution and a 0.05 M solution of sodium phosphate buffer are used as deblock solutions. The 0.10 M solution had a pH of 7.2, and such a deblock solution is used in examples demonstrating the effectiveness of synthesis on a microarray of electrodes and to show that crosstalk is prevented by using such a solution. Imaging of results is accomplished using a fluorescently labeled oligonucleotide probe, and such results show minimal crosstalk. The microarray system is such that synthesis, and hence molecular attachment, occurs on an overlayer attached to the electrode. In addition to the examples using sodium phosphate buffer, the use of acetate buffers, borate buffers, carbonate buffers, citrate buffers, HEPES buffers, MOPS buffers, phosphate buffers, TRIS buffers, and KI solutions is disclosed for use in deblocking. To contrast the effectiveness of the sodium phosphate at preventing crosstalk, Montgomery I and II provide an example using an organic deblocking solution disclosed in Southern, U.S. Pat. No. 5,667,667. The solution consisted of 1% triethylammonium sulfate in acetonitrile solvent. As shown in Montgomery I and II, the solution of Southern did not prevent crosstalk on the microarray of Montgomery I and II and showed considerable random deblocking around the area away from the active electrodes.
In contrast to Montgomery I and II, Southern disclosed the use of the acetonitrile deblock solution for use in an electrode array system having an arrangement such that synthesis, and hence deblocking, occurred on a surface opposite to the electrode surface. Unlike the array of single electrodes of approximately 90 micrometer diameter in Montgomery I and II, the array in Southern consists of linear electrodes ranging from 250 micrometers in width to 0.5 millimeters in width. An array having 50 to 100 micrometers width is disclosed as a future production model but no examples on such an array are provided. Southern demonstrated deblocking on a prepared glass slide held opposite to an electrode array, but such deblocking occurred in a line that was 200 micrometers to 0.5 millimeters in width. The larger size scale and electrode arrangement in Southern contrast with that disclosed in Montgomery I and II such that the organic deblock of Southern was not effective in the electrode arrangement and the smaller size scale of Montgomery I and II.
As a result of the examples provided in Montgomery I and II, the use of an organic deblock would not be encouraged for use on a microarray for synthesis of DNA, peptides or other polymeric materials. However, such a deblock may be useful for such synthesis where an aqueous media may need to be avoided. Thus, the development of a suitable organic deblock solution is desirable for synthesis conditions where there is a need to avoid an aqueous solution in the deblocking step. The present invention provides such an organic deblock solution for removing acid-labile protecting groups and substantially prevents crosstalk on an electrode microarray wherein synthesis occurs on an overlayer on the electrode microarray.